Tilapia Lake Virus: a Threat to the Global Tilapia Industry? Mona Dverdal Jansen1 , Ha Thanh Dong2 and Chadag Vishnumurthy Mohan3

Reviews in Aquaculture, 1–15 doi: 10.1111/raq.12254

Tilapia lake virus: a threat to the global tilapia industry? Mona Dverdal Jansen1 , Ha Thanh Dong2 and Chadag Vishnumurthy Mohan3

1 Norwegian Veterinary Institute, Oslo, Norway 2 Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi (KMUTT), Bangkok, Thailand 3 WorldFish, Penang, Malaysia

Correspondence Abstract Mona Dverdal Jansen, Norwegian Veterinary Institute, Pb 750 Sentrum, N-0106 Oslo, Tilapia lake virus (TiLV) is a recently described virus affecting wild and farmed Norway. Email: mona-dverdal.jansen@ tilapines. At present, it has been reported on three continents (Asia, Africa and vetinst.no South America) and the number of countries where the agent has been detected is likely to increase rapidly as a result of increased awareness, surveillance and avail- Received 24 January 2018; accepted 10 April ability of diagnostic methods. Any lack of openness regarding the TiLV status of a 2018. translocating live tilapia population destined for aquaculture may inadvertently contribute to the spread of the agent. Currently, there is no cure for viral diseases in aquaculture and while vaccines and selective breeding have proved successful in reducing the severity of some viral diseases, there are currently severe knowledge gaps relating to TiLV and no effective, affordable vaccines are yet available. This paper summarizes the published scientific information on TiLV and highlights important issues relating to its diagnosis, mitigation and control measures. While there have been no scientific studies on the socio-economic impact of TiLV, it may pose a significant threat particularly to small-scale fish farmers’ livelihoods and wild tilapine populations if left uncontrolled. To aid disease investigations, the authors propose case definitions for suspected and confirmed cases of TiLV infections.

Key words: review, syncytial hepatitis, tilapia, Tilapia lake virus.

ascites and abnormal behaviour (Amal & Zamri-Saad 2011; Introduction Suwannasang et al. 2014). In 1997, it was estimated that Tilapia (Oreochromis sp.) is farmed on several continents, the yearly economic loss due to infection with Streptococ- with production ranging from extensive backyard ponds to cus was in the order of $150 million (Shoemaker & Klesius large, commercial operations. According to the Food and 1997). Apart from Streptococcosis, there are several com- Agriculture Organization of the United Nations (FAO), the mon infectious diseases in farmed tilapia. Columnaris global production of tilapia was estimated at 6.4 million caused by Flavobacterium columnare often shows clinical tons (MT) in 2015, with the top three producers being the signs of necrotic gills, fin rot, skin erosion or necrotic mus- People’s Republic of China (1.78 MT), Indonesia cle (Figueiredo et al. 2005; Dong et al. 2015a). Francisel- (1.12 MT) and Egypt (0.88 MT) (FAO 2017a). Bangladesh, losis caused by Francisella noatunensis subsp. orientalis and Vietnam and the Philippines are other leading producers Edwardsiellosis caused by Edwardsiella ictaluri produce (FAO 2017a). clinical signs of visceral white spots in internal organs (Soto Amongst the advantages of farming tilapia is its general et al. 2009, 2012; Nguyen et al. 2016). Haemorrhagic septi- hardiness, adaptability to various production systems and caemia caused by motile aeromonads (Aeromonas hy- rapid growth, with advances in genetic selection and tar- drophila, A. sobria, A. veronii and A. jandaei) may present geted breeding having further improved these characteris- clinical signs of haemorrhage, exophthalmia and ascites (Li tics (Ponzoni et al. 2011; e.g. FAO 2017b). Amongst & Cai 2011; Dong et al. 2015b, 2017d) and mixed clinical important disease challenges are Streptococcus infections, signs of complicated multiple infections (Dong et al. which affect production worldwide and can occur in most 2015b; Assis et al. 2017). A vast array of viruses have been production systems. Clinical signs of streptococcal infec- reported to affect cultured finfish (Crane & Hyatt 2011; tion may include skin haemorrhages, ocular alterations, Zhang & Gui 2015). In tilapia, several viral infections were

© 2018 The Authors Reviews in Aquaculture Published by John Wiley & Sons Australia, Ltd 1 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. M. D. Jansen et al. occasionally reported in tilapia fry including betanodavirus with TiLV as tilapia lake virus disease (OIE 2017a), other and tilapia larvae encephalitis virus (TELV) which present names such as syncytial hepatitis of tilapia (SHT) (Fergu- with neurological signs of erratic swimming or whirling son et al. 2014) and 1-month mortality syndrome (Tat- syndrome (Shlapobersky et al. 2010; Keawcharoen et al. tiyapong et al. 2017) have been used in scientific papers. 2015); infectious spleen and kidney necrosis virus (ISKNV) TiLV has been identified in samples from farms experienc- associated with gross signs of lethargy, gill pallor and dis- ing summer mortalities in Egypt (Fathi et al. 2017); how- tension of the coelomic cavity (Subramaniam et al. 2016). ever, the direct association with the virus and the summer In the late 2000s, there was a large reduction in the mortality events has not yet been determined. annual wild catch of the Israeli Sea of Galilee’s main edible In addition to scientific papers and OIE notification doc- fish, S. galilaeus, from 316 metric tons in 2005 to a low of uments, several other non-scientific documents, such as a 8 metric tons in 2009 (Eyngor et al. 2014). At the same Network of Aquaculture Centres in Asia-Pacific (NACA) time (2009), large losses of farmed tilapia were recorded disease advisory (NACA 2017) and a CGIAR Research Pro- throughout Israel (Eyngor et al. 2014). A novel RNA virus gram on Fish Agri-food Systems factsheet (CGIAR 2017), was subsequently identified and termed tilapia lake virus have been published in relation to this emerging disease (TiLV) (Eyngor et al. 2014). Subsequent to the Israeli pub- problem in an effort to notify relevant stakeholders. This lication, scientific publications have reported identification review summarizes the currently available scientific infor- of TiLV from samples collected in Colombia (Kembou Tso- mation on TiLV and highlights important research gaps fack et al. 2017), Ecuador (Ferguson et al. 2014; Bacharach and issues relating to prevention and control of the associ- et al. 2016a), Egypt (Fathi et al. 2017; Nicholson et al. ated disease. Some additional information currently only 2017), India (Behera et al. 2018), Indonesia (Koesharyani available in grey literature and through personal communi- et al. 2018), Malaysia (Amal et al. 2018) and Thailand cation has also been included for the sake of completeness. (Dong et al. 2017a; Surachetpong et al. 2017). In May 2017, the FAO released a Global Information and Early Aetiological agent Warning System (GIEWs) special alert 338 on TiLV (FAO 2017c) and the World Organization for Animal Health Viral properties (OIE) published a TiLV technical disease card (OIE 2017a). The virus has been described as a novel enveloped, nega- TiLV is currently not listed by the OIE, but there is ongoing tive-sense, single-stranded RNA virus with 10 segments work evaluating whether listing should take place. How- encoding 10 proteins (Eyngor et al. 2014; Bacharach et al. ever, TiLV is listed for reporting under the NACA regional 2016a; Surachetpong et al. 2017) and a diameter between Quarterly Aquatic Animal Diseases (QAAD) reporting 55 and 100 nm (Ferguson et al. 2014; Eyngor et al. 2014; system for the Asia-Pacific. Subsequent to the OIE publica- del-Pozo et al. 2017; Surachetpong et al. 2017; Fig. 1). All tion, six countries/territories have submitted notification 10 segments contain an open reading frame (ORF), with to the OIE of TiLV presence, namely Chinese Taipei the largest segment, segment 1, containing an open reading (OIE 2017b), Israel (OIE 2017c), Thailand (OIE 2017d), frame with weak sequence homology to the influenza C Malaysia (OIE 2017e), Peru (OIE 2018) and the Philippines virus PB1 subunit (~17% amino acid identity, 37% seg- (OIE 2017f). While the OIE terms the disease associated ment coverage) (Bacharach et al. 2016a). The remaining

Figure 1 Transmission electron micrographs of TiLV-infected fish liver tissue showing cytoplasmic viral particles (white arrows) at low (a) and high (b) magnification. The electron micrograph in (b) is a magnification of the box outlined in black in (a), and the same 3 example virions (80–90 nm diameter) are indicated with white arrows in both electron micrographs (Images by H.T. Dong).

Reviews in Aquaculture, 1–15 2 © 2018 The Authors Reviews in Aquaculture Published by John Wiley & Sons Australia, Ltd Tilapia lake virus threat to tilapia industry segments show no homology to other known viruses (Eyn- epithelium (del-Pozo et al. 2017). Analyses of samples orig- gor et al. 2014; Bacharach et al. 2016a); however, the con- inating from the Tanzanian and Ugandan parts of Lake served complimentary sequences at the 50 and 30 termini Victoria found TiLV RNA prevalence to be highest in the are similar to the genome organization found in orthomyx- spleen, followed by the head kidney, heart and liver oviruses (Bacharach et al. 2016a). A taxonomic proposal (Mugimba et al. 2018). No brain samples were found to be has been submitted to the International Committee on TiLV-positive; however, only two of the 17 ﬁsh where the Taxonomy of Viruses (ICTV) for a new, unassigned genus brain was sampled tested positive for TiLV by another tis- Tilapinevirus that include the new species Tilapia tilap- sue (Mugimba et al. 2018). inevirus (Bacharach et al. 2016b). Viral particles have been found to be sensitive to organic Genetic variation solvents (ether and chloroform) due to their lipid mem- brane (Eyngor et al. 2014). Duration of survival outside the Currently, TiLV sequences from samples originating from host has not been determined; however, horizontal, water- Ecuador, Egypt, India, Israel, Malaysia, Tanzania (Lake Vic- borne spread has been demonstrated under experimental toria), Thailand, Uganda (Lake Victoria) are available in conditions (Eyngor et al. 2014). the GenBank database (https://www.ncbi.nlm.nih.gov/ge Results from in situ hybridization (ISH) indicate that nbank/). The majority of sequences are from segment 1; TiLV replication and transcription occurs at sites of pathol- however, there are currently two whole-genome sequences ogy (i.e. the liver in samples with liver lesions and the cen- available, one from Israel (Bacharach et al. 2016a) and one tral nervous system in samples with central nervous system from Thailand (Surachetpong et al. 2017). lesions) (Bacharach et al. 2016a). In samples collected from The reported nucleotide identity between the Israeli TiLV Thailand, ISH yielded positive signals in multiple organs (prototype strain) and isolates originating from South (liver, kidney, brain, gills, spleen and muscle connective tis- America, Africa and Asia has been listed in Table 1. From sue), with the strongest signals found in liver, kidney and samples originating in Israel, available sequences include gills (Dong et al. 2017a). In samples originating from Ecua- KJ605629 (clone 7450, ORF) (Eyngor et al. 2014) and dor, a viral predilection to liver and gastrointestinal tract KU751814 to KU751823 (whole genome, segments 1–10) was suggested, with an apparent tropism for hepatic (Bacharach et al. 2016a) and KU552132 (Tal et al. 2016).

Table 1 Overview of available TiLV sequences in GenBank and the percentage nucleotide identity found between sequences originating from Israel and sequences from other countries and territories

Source (non-Israeli GenBank accession Identity to TiLV from Israel (prototype strain) References sources) no. GenBank accession no. of Israeli TiLV % nt identity

Chinese Taipei Not available Segment 3 (Accession number not specified) 93% OIE (2017b) Ecuador Not available Full genome sequences 97.2–99.0% Bacharach et al. (2016a) KU751814–KU751823 Ecuador Not available KJ605629 (ORF) 98% to 100% del-Pozo et al. (2017) Egypt Not available KU751816 (segment 3) 93% Fathi et al. (2017) Egypt KY817381–KY817390 Segments 3, 4 and 9 (Accession numbers 93% Nicholson et al. (2017) not specified) India MF502419, MF574205 KJ605629 (segment 3) 96.4–97.2% Behera et al. (2018) and MF582636 Indonesia Not available KU751816 and KJ605629 (segment 3) 97% Koesharyani et al. (2018) Malaysia MF685337 KU751822 (segment 9) 97% Amal et al. (2018) Philippines Not available Segment 3 (Accession number not specified) 94–95% OIE (2017f) Tanzania (Lake MF526980–MF526996 KU552132 (contig 7 = segment 2) Not given† Mugimba et al. (2018) Victoria) KU751815 (= NC029921, segment 2) Thailand KY615742 KU751814 (segment 1) 96.3–97.5% Dong et al. (2017a) Thailand KY615743 KU751818 (segment 5) Thailand KY615744 to KY615745 KU751822 (segment 9) Thailand KX631921 Full genome sequences KU751814–KU751823 95.6–99.1% Surachetpong et al. (2017) KX631930–KX631936 Uganda (Lake MF536423–MF536432 KU552132 (contig 7 = segment 2) Not given† Mugimba et al. (2018) Victoria) KU751815 (= NC029921,segment 2)

†Authors state that sequences were ‘identical with’ or ‘closely related to’ the Israeli sequences.

Host factors Clinical signs and diagnostics Susceptible species Clinical signs and gross pathology Affected farmed species include hybrid tilapia (Oreochromis The reported clinical signs and gross pathological lesions niloticus 9 O. aureus hybrids) in Israel (Eyngor et al. associated with TiLV infections are somewhat variable, 2014); Nile tilapia (O. niloticus) in Ecuador (Ferguson depending on geographical origin. Clinical signs include et al. 2014), Egypt (Fathi et al. 2017), India (Behera et al. lethargy, ocular alterations, skin erosions and discoloration 2018), Indonesia (Koesharyani et al. 2018), Thailand (darkening) in farmed tilapia in Israel (Eyngor et al. 2014) (Dong et al. 2017a; Surachetpong et al. 2017) and Uganda while lethargy, skin erosions and ocular lesions were (Mugimba et al. 2018); red tilapia (Oreochromis sp.) in reported from cases in wild tilapines (OIE 2017c). The case Thailand (Dong et al. 2017a; Surachetpong et al. 2017) and in Ecuador presented with exophthalmia, discoloration red hybrid tilapia (Oreochromis niloticus 9 O. mossambi- (darkening), abdominal distension, scale protrusion and cus) in Malaysia (Amal et al. 2018). gill pallor (Ferguson et al. 2014), while ulcers and exoph- A range of wild tilapines (including Sarotherodon galilaeus, thalmia have been reported from Peru (OIE 2018). In Thai- Tilapiazilli, Oreochromis aureus,andTristamellasimonis land, loss of appetite, lethargy, abnormal behaviour (e.g. intermedia) from the Sea of Galilee have tested positive for swimming at the surface, stop schooling), pallor, anaemia, TiLV in Israel (Eyngor et al. 2014). In Malaysia, wild black exophthalmia, abdominal swelling and skin congestion and tilapia (Oreochromis sp.) has been reported affected (OIE erosion have been reported (Dong et al. 2017a; Surachet- 2017e), while wild Nile tilapia tested positive in Lake Victoria pong et al. 2017). Brain congestion and paleness of the gills (Tanzania and Uganda) (Mugimba et al. 2018) and in Peru and liver have additionally been observed (Surachetpong (OIE 2018). Upon testing in Malaysia, Tinfoil barb (Pun- et al. 2017). In the laboratory, naturally infected Thai Nile tius schwanenfeldii) was found to be TiLV-positive; however, tilapia fingerlings showed darkening and some moribund the significance of this remains yet to be determined (Azila fish exhibited scale protrusion prior to death (Dong, per- Abdullah, personal communication). sonal observation). From India, clinical signs in naturally Cocultivated grey mullet (Mugil cephalus) and carp infected fish were skin erosions and loss of scales while (Cyprinus carpio) have not shown mortality during disease experimentally infected fish exhibited exophthalmia, swol- outbreaks in Israel (Eyngor et al. 2014). Similarly, coculti- len abdomen and scale protrusion (Behera et al. 2018). vated grey mullet and thin-lipped mullet (Liza ramada) Clinical signs reported from the Philippines include were found to be unaffected during Egyptian outbreaks abdominal swelling and bulging of the eyes (OIE 2017f). In (Fathi et al. 2017) and cocultivated Indian Major Carps Egyptian farms experiencing ‘summer mortality’, affected (rohu (Labeo rohita), catla (Catla Catla), mrigal (Cirrhi- fish showed haemorrhagic patches, detached scales, open nus mrigala)), milk fish (Chanos chanos) and pearl spot wounds, dark discoloration and fin rot, with some of these (Etroplus suratensis) were unaffected in India (Behera et al. fish testing positive for TiLV, with or without coinfection 2018). by Aeromonas spp. (Nicholson et al. 2017). No case description was provided for the fish from which the Colombian samples originated (Kembou Tsofack et al. Susceptible life stages 2017). A range of clinical signs representative of TiLV infec- In Israel, mortalities have been observed over a wide weight tion is shown in Figure 2. Based on the available informa- range (Eyngor et al. 2014). Fingerlings and juveniles (up to tion, it seems that a complete list of pathognomonic signs, 80 g) have been affected in Ecuador (Ferguson et al. 2014), to allow a reliable diagnosis based on clinical signs alone, is India (Behera et al. 2018), Malaysia (Amal et al. 2018) and not currently feasible. Thailand (Dong et al. 2017a; Surachetpong et al. 2017). In Egypt, medium- (>100 g) and large-sized fish have been Histopathology affected by summer mortality, some of which have tested positive for TiLV (Fathi et al. 2017). Both juvenile and There appear to be some geographical and individual varia- adult tilapia have been reported affected in Peru (OIE tions in the histopathological lesions associated with TiLV 2018). Early developmental stages of tilapia (fertilized eggs, infections. Observed lesions in affected fish in Israel include yolk-sac fish and fry) have also tested positive for TiLV congestion of internal organs (kidney and brain), foci of (Dong et al. 2017b). Subclinical TiLV infection has been gliosis and perivascular cuffing in the brain cortex and ocu- reported from Thailand in clinically healthy adults (two of lar lesions (endophthalmitis and cataractous changes of the two tested) and fingerlings (nine of 19 tested), with no clin- lens) (Eyngor et al. 2014). In affected fish from Egypt, ical disease reported to have been observed 1 month after histopathological findings included gliosis, encephalitis and sampling was completed (Senapin et al. 2018). mild perivascular cuffing in the brain, multifocal chronic

(a) (c)

(b) (d)

Figure 2 Clinical signs of representative TiLV-infected Nile tilapia and red tilapia: naturally diseased Nile tilapia showing discolouration, loss of scales and skin erosion (a), naturally diseased red tilapia showing skin haemorrhages (b), experimentally diseased Nile tilapia showing exophthalmia, abdominal swelling and scale protrusion (c and d). Images (a), (c) and (d) are reprinted from Aquaculture, Volume 484, Behera et al., Emergence of tilapia lake virus associated with mortalities of farmed Nile tilapia Oreochromis niloticus (Linnaeus 1758) in India, pages 168–174, Copyright (2018), with permission from Elsevier. Image C provided by H. T. Dong (taken in conjunction with the outbreak described in Dong et al. 2017a.).

hepatitis and multifocal interstitial haemorrhage in the kid- hepatitis to be the most common histopathological feature ney (Fathi et al. 2017). The case in Ecuador showed hepa- found in TiLV outbreaks. While it was not reported from tocyte necrosis and syncytial cell formation, necrosis of outbreaks in the earliest report from Israel (Eyngor et al. gastric glands and diffuse congestion in multiple tissues 2014), syncytial hepatitis was described in a later study (Ferguson et al. 2014). Syncytial hepatitis was also reported from the same research group (Bacharach et al. 2016a). in samples from Colombia (Kembou Tsofack et al. 2017), India (Behera et al. 2018), Malaysia (Amal et al. 2018) and Cell culture Thailand (Dong et al. 2017a). In Indian cases, syncytial cell formation was observed in the liver of both naturally and Experiments have shown multiple cell lines to be suitable experimentally infected fish and occasionally observed in for TiLV cell culture (Eyngor et al. 2014; Kembou Tsofack the brain of experimental fish (Behera et al. 2018). Addi- et al. 2017). tional observations from Thailand include aggregation of The E-11 cell line was found to show visible cytopathic lymphocytes and perivascular cuffing in brain tissue (Sura- effect (CPE) 5–7 days after inoculation, with cytoplasmic chetpong et al. 2017). The presence of eosinophilic intracy- vacuoles and plaque formation followed by disintegration toplasmic inclusions has been described with both natural of cell monolayer nine to 10 days after inoculation (Eyngor infections (Ferguson et al. 2014) and in experimentally et al. 2014; Tattiyapong et al. 2017). Cell lines of primary infected fish (Tattiyapong et al. 2017). tilapia brain cells showed swollen, rounded, granulated cells A degree of histopathological variation in fish from a sin- 10–12 days after inoculation, with monolayer detachment gle farm has been observed in Thailand (Fig. 3). While syn- 14–19 days after inoculation (Eyngor et al. 2014). E-11 cytial hepatitis and foamy cytoplasm were observed in the cells at 25°C have been reported to provide optimal condi- liver of the majority of tested fish, all naturally infected fish tions for TiLV replication (Kembou Tsofack et al. 2017). showed severe pancreatic necrosis and some occasionally The OmB and TmB cell lines derived from O. mossambicus exhibited the presence of intracytoplasmic inclusion bodies showed similar sensitivities to TiLV infection as the E-11 in the hepatocytes. Inflammation with severe infiltration of cell line; however, the E-11 cultures were deemed superior lymphocytes was observed in some areas of the kidney due to the clear and rapid CPE development (Kembou Tso- tubules and brain where syncytial cells were located in the fack et al. 2017). OmB has been suggested as a useful cell centre of areas of inflammation (Dong, personal observa- line for endpoint dilution (TCID50) assays and both OmB tion). Currently available information suggests syncytial and TmB may reportedly be useful for generating pure

Figure 3 Photomicrographs of haematoxylin and eosin-stained sections of tissue from the liver, kidney and brain of normal fish (a, e, g) and TiLV- infected fish (b–d, f, h). The infected liver tissue showed syncytial hepatocytes and foamy cytoplasm (b), intracytoplasmic inclusion bodies (c) and inflammation with pancreatic necrosis (d). Kidney tissue showed syncytial cells and severe infiltration of inflammatory lymphocytes (f). Brain tissue also showed syncytial cells and severe infiltration of inflammatory lymphocytes (h) (Images by H.T. Dong.)

Figure 4 Coding strand sequence of TiLV genome segment 3 (KU751816). Putative start and stop codons are enclosed in boxes. The shaded sequences represent positions of primers used in nested PCR (from left to right; Nested ext-2, ME1, 7450/150R/ME2 and Nested ext-1) published by Eyngor et al. (2014) and Kembou Tsofack et al. (2017). The two inner primers were also employed for SYBR green-based RT-qPCR (Kembou Tsofack et al. 2017). Nested ext-2 primer indicated by shaded underlined sequence was omitted in the semi-nested RT-PCR method (Dong et al. 2017a). Double underlines indicate the sites that were used to design primers for a recent SYBR green-based RT-qPCR protocol (Tattiyapong et al. 2018).

TiLV strains as they are snakehead reovirus-free (Kembou PCR assay using the same primer sets with detailed condi- Tsofack et al. 2017). Pooling of two to three samples of tions enabling detection down to seven copies of TiLV, brain tissue has yielded positive TiLV culture results (Kem- 10 000 times more sensitive than the single RT-PCR (limit bou Tsofack et al. 2017). detection of ~70 000 copies) (Kembou Tsofack et al. 2017). More recently, the CFF cell line, originating from The nested RT-PCR assay was found to detect TiLV in both Pristolepis fasciatus, has been used for viral propagation in fresh and preserved (RNAlater; QIAGEN) samples from India, which exhibited CPE at day 3 postinoculation and diseased fish, and identified TiLV RNA in samples from caused severe cell detachment at days 6–7 postinfection diseased fish from Israel, Ecuador and Colombia (Kembou (Behera et al. 2018). Most recently, two tilapia-derived cell Tsofack et al. 2017). A SYBR green-based qPCR method lines, OnlB from brain and OnlL from liver, has been using nested primers (ME1 & 7450/150R/ME2) gained a shown to be highly permissive for propagating TiLV limit detection of 70 copies (Kembou Tsofack et al. 2017). (Swaminathan et al. 2018). Other cell lines (CHSE-214, Subsequently, an alternative semi-nested RT-PCR method BF-2, BB, EPC, KF-1, RTG-2 and FHM) have been reported has been described where the primer Nested ext-2 was to show inconsistent CPE with TiLV (Eyngor et al. 2014). omitted to reduce the risk of false-positive detections No CPE was reported in CCKF, RTF, PSF, HBF and FtGF (Dong et al. 2017a). This protocol had a detection limit of cells lines (Swaminathan et al. 2018). 7.5 copies (Dong et al. 2017a) and was able to detect TiLV from clinically healthy fish (Senapin et al. 2018). Pooling of between two to five samples has been reported to yield suc- Molecular methods cessful agent identification (Dong et al. 2017a; Fathi et al. Several polymerase chain reaction (PCR)-based methods 2017; Kembou Tsofack et al. 2017; Surachetpong et al. for TiLV detection have been described in the literature. 2017). Recently, a newly SYBR green-based reverse tran- Initially, a reverse transcriptase-PCR (RT-PCR) method for scription quantitative PCR (RT-qPCR) method targeting TiLV detection was published together with information the same genome segment 3 was developed for detection of on TiLV-specific primers targeting segment 3 (Eyngor et al. TiLV from clinical samples with a reported sensitivity 2014). This was followed by the publication of a nested RT- of two copies/lL (Tattiyapong et al. 2018). The locations

Reviews in Aquaculture, 1–15 © 2018 The Authors Reviews in Aquaculture Published by John Wiley & Sons Australia, Ltd 7 M. D. Jansen et al. of the published primers are shown in Figure 4. None of tilapia production, namely Africa (Egypt (e.g. Fathi et al. the currently available PCR methods have been fully vali- 2017), Tanzania (Mugimba et al. 2018) and Uganda dated. Until validations have been performed and pub- (Mugimba et al. 2018)), Asia (Chinese Taipei (OIE 2017b), lished current detection methods should be combined with India (Behera et al. 2018), Indonesia (Koesharyani et al. sequencing of representative PCR products for agent con- 2018), Israel (e.g. Eyngor et al. 2014; OIE 2017c), Thailand firmation. (e.g. Dong et al. 2017a; OIE 2017d), Malaysia (OIE 2017e; An in situ hybridization (ISH) method has been described Amal et al. 2018) and the Philippines (OIE 2017f) and South and has been used to reveal TiLV tissue tropism (Bacharach America (Colombia (Kembou Tsofack et al. 2017), Ecuador et al. 2016a; Dong et al. 2017a). ISH revealed that mRNA of (e.g. Bacharach et al. 2016a) and Peru (OIE 2018)). the virus was detected in both the nucleus and the cytoplasm The majority of countries have reported a limited num- of the infected cells (Bacharach et al. 2016a). It should be ber of TiLV detections and/or disease outbreaks so far. The noted that ISH using a DIG-labelled probe derived from exception is Israel where TiLV has been found more wide- the partial genome segment 3 of TiLV (415 bp) allowed spread, ranging from wild tilapia stocks in the Sea of Galilee detection of TiLV from different organs of naturally to farmed stocks in all the major aquaculture areas (coastal TiLV-infected fish which tested positive in the first step RT- shore, Jordan Valley and Upper and Lower Galilee) (Eyn- PCR (Dong et al. 2017a). However, no detectable positive gor et al. 2014). In Egypt, 37% of randomly selected fish signal by ISH was found for those samples that tested posi- farms in the major aquaculture areas (Kafr el Sheikh, tive for TiLV in the second step RT-PCR, suggesting that Behera, Sharkia) were affected by summer mortalities when the ISH method by Dong et al. (2017a) may be suitable for sampled in 2015 (Fathi et al. 2017). Amongst sampled heavily infected samples only (Dong, personal observation). farms that had experienced ‘summer mortalities’, four of eight farms sampled in 2015 (Nicholson et al. 2017) and three of seven farms sampled in 2016 (Fathi et al. 2017) Serology were found to be TiLV-positive. There are no publications relating to the use of serology in the Although TiLV became known to science in 2014, the context of TiLV diagnostics. Ferguson et al. (2014) observed virus was suspected to be responsible for massive mortali- a reduced packed cell volume (16% versus the normal ties of tilapia in Israel and Ecuador since 2008–2009 (Eyn- 48–50%) in the TiLV case in Ecuador, together with an gor et al. 2014; Bacharach et al. 2016a; FAO 2017c). increased number of immature erythrocytes in blood smears. Similarly, TiLV was reported in Thailand in early 2017 (Dong et al. 2017a; Surachetpong et al. 2017). However, samples collected in 2015 and 2016 (Surachetpong et al. Nonlethal sampling 2017), and archived samples from unexplained mortalities A comparative study of mucus and liver samples collected events between 2012 and 2016 (Dong et al. 2017b), have from 35 randomly selected moribund and healthy adult tested positive for TiLV. A SHT histopathological feature red tilapia revealed identical TiLV status for both tissues resembling TiLV infection was observed in the experimen- when analysed by the RT-qPCR method described by tal fish used for a student thesis (MSc) at Chulalongkorn Tattiyapong et al. (2018) (Liamnimitr et al. 2018). Twenty- University in 2014 (Weerapornprasit et al. 2014), possibly one of 35 samples tested positive for TiLV, with the Ct representing an earliest known case of SHT in Thailand values for the two tissue types noted to be relatively close (Nopadon Pirarat, personal communication). Retrospec- (Liamnimitr et al. 2018), although no statistical evaluation tive investigation of available archived samples in other fish was reported. TiLV-infected mucus inoculated onto E-11 health laboratories may shed further light on origins and cells showed typical CPE after 3–7 days after infection distribution of TiLV (Dong et al. 2017b). (Liamnimitr et al. 2018). In an infection trail, TiLV RNA was detected in cohabitant fish mucus one to 12 days Mortality in natural and experimental outbreaks postinfection (dpi) (comparative values for liver and intesti- nes being two to 14 dpi and five to 12 dpi, respectively, with High levels of mortality have been reported in association no TiLV was detected in faeces) (Liamnimitr et al. 2018). with TiLV infections on all three continents. Mortality levels of above 80% have been observed in affected farmed populations in Israel, while no such level of mass mortality Epidemiology has been reported in wild stocks from which positive sam- Geographical distribution ples have been obtained (Eyngor et al. 2014). In Thailand, Based on information in scientific publications, OIE notifica- mortality levels between 20% and 90% have been reported, tions and sequence information available in Genbank, TiLV with mortality usually seen within the first month after appears to be present on the three continents with the largest transfer to grow-out cages (Dong et al. 2017a;

Surachetpong et al. 2017) and peak mortality rates stocking density, fry weight at transfer, weather pattern, observed within 2 weeks of onset of mortality (Surachet- water temperature, dissolved oxygen, the number of days pong et al. 2017). Similarly, a case in Ecuador showed onset spent preparing the pregrow-out pond, month of transfer of mortality from 4 to 7 days posttransfer to on-growing to pregrow-out pond, daily feeding rate, number of pond ponds, with mortality ranging from a low level of 10–20% production cycles per year, year of stocking and mortality to a high level of 80%, depending on the fish strain (Fergu- rate per production cycle (Kabuusu et al. 2018). It was son et al. 2014). In India, outbreaks of TiLV associated found that infected populations showed about five times with 80–90% mortality (Behera et al. 2018). In contrast, higher mortality levels than uninfected populations the average mortality level at farms experiencing ‘summer (RR = 4.8, 95% CI 2.9–7.9), with tilapia of the Chitralada mortality’ in Egypt was found to be 9.2% (range 5–15%), strain showing twice as high mortality as GMT and GIFT with the mortality level attributable to TiLV infection cur- strains (RR = 2.1, 95% CI 1.8–2.4) (Kabuusu et al. 2018). rently unknown (Fathi et al. 2017). Similarly, several cases Excess mortality was significantly associated with dissolved of natural TiLV infection have been found to be associated oxygen, stocking density (fish/m2), number of pond pro- with relatively low levels of mortality (6.4% in Chinese Tai- duction cycles per year (Kabuusu et al. 2018). In Egypt, pei and 0.71% and 15% in wild and farmed tilapia, respec- large farm size, high stocking densities and tilapia-mullet tively, in Malaysia) (OIE 2017b,e). Subclinical infections in polyculture have been identified as risk factors for TiLV both adults and fingerlings have also been reported in Thai- outbreaks (Fathi et al. 2017). land (Senapin et al. 2018), and variations in mortality have In Ecuador, increased water temperature (°C) and been reported in farms with different species and produc- increased fry weight at transfer (g) were protective factors tion form combinations in Thailand, ranging from around for both excess mortality and severe SHT (defined as very 20% in farms with mixed stocking of red tilapia and Nile high excess mortality), and no association was found tilapia in earthen pond in Phetchaburi province to around between season (wet/dry) and the presence and severity of 90% in farms with Nile tilapia in Pathum Thani province SHT (Kabuusu et al. 2018). Clinical outbreaks have been and farms with red tilapia in open floating cages in Chai reported during the hot season, namely May to October (at Nat province (Dong et al. 2017a). A significant strain dif- water temperatures of 22°Cto32°C) in Israel (Eyngor et al. ferences in mortality have been observed in Ecuador where 2014), June to October (≥25°C) in Egypt (Fathi et al. 2017) the Chitralada strain was found to have significantly higher and May to November (25°Cto27°C) in Ecuador (Fergu- mortality than GMT and GIFT strains (Ferguson et al. son et al. 2014). Some of the samples yielding positive TiLV 2014; Kabuusu et al. 2018). detection in Thailand were collected in the months between Successful viral propagation has allowed the conduction October and May (Surachetpong et al. 2017). Affected fin- of TiLV infection experiments by several research groups. gerlings in Ecuador were commonly detected within 4– In Israel, experimental infection of Nile tilapia juveniles 7 days posttransfer to grow-out ponds (Ferguson et al. (30–35 g, strain Chitralada) by intraperitoneal (IP) injec- 2014), with tilapia strain being found to be a risk factor for tion resulted in 75–85% mortality within 10 days, with a high mortality (Ferguson et al. 2014; Kabuusu et al. 2018). similar mortality pattern observed in a cohabitation experi- In Thailand, variations in mortality have been observed ment (Eyngor et al. 2014). In Thailand, recorded mortality with different species and production form combinations levels of red tilapa and Nile tilapia juveniles (~30 g) within (Dong et al. 2017a). 12 dpi were 66% and 88%, respectively (Tattiyapong et al. 2017), with a second cohabitation trail showing a cumula- Coinfections tive mortality level of 55.7% for cohabitant fish (Liamnim- itr et al. 2018). A cumulative mortality of 100% by day It has previously been proposed that multiple infections seven postinfection was observed in an infection trail using outweigh single infection during disease outbreaks in IP injection of 12–15 g tilapia (Behera et al. 2018). Eyngor farmed tilapia (Dong et al. 2015b). In the case of TiLV, et al. (2014) noted that fish surviving disease outbreaks reported coinfections in TiLV-positive fish from Thailand have been found to be resistant to subsequent outbreaks, included bacteria (Flavobacterium, Aeromonas and Strep- which indicate a host immune response against primary tococcus), external monogenean parasites (Gyrodactylus infection suggesting that vaccination may be an appropriate and Dactylogyrus) and ciliated protozoa (Trichodina) approach for disease control. (Surachetpong et al. 2017). Several of the TiLV-positive fish from Egypt in 2015 were reported to have a coinfection of one or more Aeromonas spp. (A. veronii, A. ichthiosmia, Risk factors A. enteropelogenes and A. hydrophilia) (Nicholson et al. A study of production-level risk factors for the presence 2017). A case of coinfection between A. veronii and TiLV in and severity of SHT in Ecuador assessed tilapia strain, juvenile hybrid red tilapia (O. niloticus 9 O. mossambicus)

Reviews in Aquaculture, 1–15 © 2018 The Authors Reviews in Aquaculture Published by John Wiley & Sons Australia, Ltd 9 M. D. Jansen et al. has been reported in Malaysia, which resulted in a mortality present in over 90 countries (De Silva et al. 2004). Begin- rate of approximately 25% (Amal et al. 2018). Examination ning with the programme in 1988, GIFT represents the first of 20 diseased fish revealed an infection rate of 20% and systematic collection and transfer of Nile tilapia germplasm 50% for TiLV and A. veronii, respectively (Amal et al. from Africa to South-East Asia (Gupta & Acosta 2004; 2018). In a case of TiLV infection in red tilapia juveniles, it Acosta & Gupta 2010); however, tilapia was widely moved was observed that while all clinically diseased fish tested around also prior to this programme. As a result, interna- positive for TiLV, 50% of the examined fish were also tional trade may have been circulating the agent worldwide infected by an unknown microsporidian-like organism in through movement of live fish for aquaculture in the their muscle (Dong, personal observation). The relative absence of knowledge of the existence of an associated risk. importance of TiLV and any coinfections in terms of clini- The observation of subclinical infections increases the risk cal severity, mortality, incubation time and so on has not of such transfers having occurred. It is speculated that over been determined. 40 countries may have a theoretical risk of inadvertent TiLV introduction due to trade and suggest the importance of initiation of surveillance activities in these countries Socio-economic impact (Dong et al. 2017b,c). Similarly, extensive trade of orna- No estimate has been published on the socio-economic mental cichlids could theoretically pose a threat for the impact of TiLV in a national or global context. High levels spread of TiLV although no TiLV has been reported in of mortality have been reported from field cases on all three ornamental cichlids to date. Emergence and spread of viral continents (Eyngor et al. 2014; Ferguson et al. 2014; Dong diseases in farmed shrimp and its association with global et al. 2017a; Surachetpong et al. 2017; Behera et al. 2018) movement of live shrimp are well documented (Walker & suggesting that the impact may be significant. However, Mohan 2009). Taking cues from shrimp, it would be worth relatively lower mortality levels have been reported in some exploring if a massive ecological shift and changes in the field cases (Fathi et al. 2017; OIE 2017b,e) and subclinical trade patterns that have accompanied the establishment infections have been described (Senapin et al. 2018) and and growth of tilapia farming industry has anything to do the reasons for these differences are yet unknown. Estimates with the emergence of TiLV. from Egypt indicate a production loss of 98 000 metric As the agent was new to science until first published in tons, at a value of around USD 100 million, due to the 2014 and no easily available diagnostic tests for its detection ‘summer mortality’ syndrome in 2015 (Fathi et al. 2017), was available, it is only recently that competent authorities, of which TiLV may play a part. scientists and other stakeholders could initiate the work of The impact on wild stocks may also be highly significant, unravelling the true distribution of TiLV. With a multicon- both in economic terms and in relation to biodiversity- and tinent presence of TiLV, there is a need for regional capac- ecological effects. In the Sea of Galilee in Israel, the annual ity building within all stakeholder groups and participatory wild catch figures for the main edible fish in the lake, approaches at all stakeholder levels should be encouraged. S. galilaeus, were reported to have decreased from 316 met- According to the FAO, multiple countries have initiated ric tons in 2005 to 8 metric tons in 2009, with a subsequent official screening and surveillance programmes (FAO increase to 160 metric tons in 2013 and 140 metric tons in 2017c) and expansion of such activities in other at-risk 2014 (Eyngor et al. 2014). The contribution of TiLV infec- countries should be encouraged. The FAO has also pub- tion to this decline has not been determined; however, lished recommended biosecurity measures that countries TiLV has been identified in samples from several wild tilap- need to follow when translocating live tilapias, for countries ines, including S. Galileaus (Eyngor et al. 2014). found positive for TiLV and for countries with an unknown TiLV status (FAO 2017c). International collaboration on such screening/surveillance efforts may expedite Discussion knowledge generation while local diagnostic capacity build- Knowledge of the geographical distribution of TiLV has ing is being undertaken. Conducting TiLV import risk rapidly increased with the heightened awareness of this new analysis should be encouraged in countries with significant agent affecting tilapia. The increase in the number of coun- tilapia production where TiLV has not been detected. The tries detecting TiLV in their tilapia populations and the current uncertainty regarding the distribution of TiLV both detection of TiLV from archived samples suggest that TiLV geographically (continents and countries), and more specif- has been present as a hidden pathogen for several years. ically within the various sectors of the affected tilapia International trade of tilapia has taken place for more than industries, represents large challenges for designing and 50 years with a resultant global distribution only exceeded conducting cost-effective surveillance programmes. Added by common carp. Tilapia is native to Africa, with move- to this challenge is the need for the development of suffi- ments having taken place since 1944, and it is currently cient diagnostic capacity specific to TiLV and the

Reviews in Aquaculture, 1–15 10 © 2018 The Authors Reviews in Aquaculture Published by John Wiley & Sons Australia, Ltd Tilapia lake virus threat to tilapia industry development of a common understanding amongst all At present, none of the currently available PCR methods stakeholders. Collaborative programmes between the pri- have been fully validated. While there has been some data vate sector and relevant governments should be promoted published on the analytical- and diagnostic sensitivities and to limit the impact of TiLV and the associated disease. The specificities, none of the methods have been fully validated potential to use a nonlethal sampling for initial screening in both healthy and diseased populations and no informa- may significantly aid farmer compliance for investigation tion on optimal tissues and the appropriateness of pooling for TiLV. Nonlethal sampling methods are not new to has been made available. Therefore, it seems prudent to aquatic disease investigations. A nonlethal sampling tech- encourage the combination of the currently available PCR nique (pectoral fin) has for example been described for detection methods with sequencing of representative PCR infectious pancreatic necrosis (IPN) in salmonids (Bowers products for agent confirmation until the diagnostic tests et al. 2008), and studies on ISAV have shown early replica- have been sufficiently validated. The development of new tion in several mucosal tissues including gills, pectoral fin, or improved diagnostic methods for TiLV (e.g. enzyme- skin and gastrointestinal tract (Aamelfot et al. 2015) sup- linked immune sorbent assay (ELISA), rapid antigen strip porting the feasibility of using mucosal samples for initial test, recombinase polymerase amplification (RPA), loop- detection of TiLV. mediated isothermal amplification (LAMP)) should be The importance of an appropriate case definition, encouraged. As tilapia is frequently farmed by lower despite the presence of some knowledge gaps, has been income farmers, there is a need for low cost, accurate diag- discussed by for example Baldock et al. (2005). While nostic methods that does not require extensive laboratory Koch’s postulates have been fulfilled for TiLV by indepen- facilities. dent studies (Eyngor et al. 2014; Tattiyapong et al. 2017; There is little scientific knowledge available regarding Behera et al. 2018), the associated clinical signs and important epidemiological aspects of TiLV. For example, histopathological changes appear to reflect a degree of the reasons for the large variations in observed mortality, geographical and individual variations. As a result, no sci- which may be related to genetic variation in the virus, dif- entifically sound set of pathognomonic signs has been fering host susceptibility, environmental factors, coinfec- defined for the disease associated with TiLV infection. tions or a combination of these, need to be elucidated. Despite this fact, the authors would like to suggest some Further knowledge on viral properties such as survival of temporary case definitions for TiLV infection until definite TiLV outside the host (in water, on fomites, in fresh/frozen case definitions can be proposed. Although bound to be products), risk factors for disease outbreaks and the pres- inaccurate in some cases, it may serve as a guideline to aid ence of any nontilapine hosts/carrier species need to be fur- disease investigations, particularly in areas where TiLV has ther investigated. The survival of viral particles in general not yet been confirmed or reported. In the event of listing fish commodities has been widely discussed, but little scien- by the OIE, it will be ‘infection by TiLV’ that will be listed tific information is available. From Norway, it has been and not the resultant clinical disease. As a result, the sug- observed that infectious viral particles of infectious salmon gested case definitions are reflecting this and are also refer- anaemia (ISA) virus have been cultured after more than ring to the group of animals (e.g. pond-level), and all life 20 years in 80°C (Knut Falk, personal communication); stages of tilapia should be considered at risk and suscepti- however, its relevance for agent spread in the field remains ble. A suspected case of TiLV infection may have one or undetermined and may be questionable. Due to varying more of the following characteristics: (i) A pond/cage of factors, such as production methods and fish genetics, the tilapia fingerlings or juveniles (<80 g) with increased possibility of a variation in risk factors between different abnormal mortality during early period of cultivation (1– countries/regions should not be overlooked. Given the 4 weeks after stocking) in the absence of obvious nonin- long-term efforts that have been invested in producing fectious causes or (ii) A pond/cage of tilapia subadults/ genetically improved strains of tilapia, susceptibilities for adults with increased abnormal mortality in the absence of TiLV need to be thoroughly investigated under field condi- obvious noninfectious causes or (iii) A pond/cage where tions. Similarly, there is an urgent need to determine the the tilapia show one or more of the following clinical potential for vertical transmission of TiLV, as well as fur- signs: behavioural changes, exophthalmia or other ocular ther investigations of the frequency and duration of sub- lesions, skin erosions, discolouration, skin haemorrhage, clinical cases. Both descriptive, observational and scale protrusion and/or abdominal swelling or (iv) A experimental studies should be conducted to address such pond/cage where at least one tested tilapia show knowledge gaps. While it has been observed that fish sur- histopathological feature of syncytial hepatitis. A con- viving the initial outbreak are immune to subsequent out- firmed case of TiLV infection has a positive PCR analysis breaks (Eyngor et al. 2014), further investigations should for TiLV with subsequent sequencing of the representative be conducted before such fish are automatically assumed to PCR product showing TiLV presence. be TiLV-resistant and used as broodstock.

To minimize the impact of TiLV in affected countries spread of TiLV. However, the advantage of stocking such and reduce the risk of further spread, implementation of SPF fish would be removed if the water body to which they good biosecurity practices, combined with the introduction are transferred are inadequately cleaned and disinfected of intervention and containment programmes, should be between production cycles or there are a lack biosecurity conducted. While stringent biosecurity may be impossible measures to prevent the introduction of TiLV after stock- to achieve in the field in many tilapia operations, achieving ing. While an SPF programme, combined with early the highest possible biosecurity standards for the operation immersion/oral vaccination prior to stocking, would be an in question should be sought. The importance of biosecu- ideal approach for effective prevention of not only TiLV rity measures needs to be promoted by competent authori- but also other major diseases in farmed tilapia, it may be ties as there is currently no cure for viral diseases in unrealistic for the majority of small-scale tilapia producers. aquaculture. The combination of biosecurity measures, While the initial proposal was to classify this virus as an breeding of fish with improved genetic resistance and vacci- Orthomyxo-like virus based on the genome arrangement nation have proven useful in reducing the number of viral (Bacharach et al. 2016a), the fact that only segment 1 shows disease outbreaks in some cases (e.g. IPN in Norwegian sal- a weak homology to PB of influenza virus is not fully sup- monid production (Hjeltnes et al. 2017). A patent for a porting this classification. The frequently observed TiLV-vaccine has been filed in the United States (Anony- histopathological feature of SHT has never been reported mous, 2017), and vaccination against TiLV may be a reality related to Orthomyxovirus but do occur in outbreaks in the future. However, it remains to be seen whether such caused by Paramyxovirus, a nonsegmented RNA virus a vaccine will be sufficiently cost-effective and easily admin- (Phillips et al. 1991; Sussman et al. 1994). Thus, in terms istered to allow a widespread dissemination and use in a of genomic characterization, the proposed Tilapia Tilap- large proportion of the major tilapia-producing countries. inevirus, in a new genus Tilapinesvirus as proposed by It is a general concern that vaccinated fish may test positive Bacharach et al. (2016b), seems more appropriate. There by PCR analysis for the agent against which they have been are, however, important aspects in terms of taxonomy, gen- vaccinated. This may be of particular concern in cases ome and protein functions, receptors and so on that needs where there has been an intraperitoneal vaccination with to be studied. For ISA, both virulent and avirulent strains subsequent sampling of abdominal tissues for diagnostic of the virus have been identified (Christiansen et al. 2011; testing. Any proportion of false positives due to vaccine Cottet et al. 2011) and alterations in virulence and cell residues is likely to be of special concern for tilapia export- tropism have been observed in the field (Christiansen et al. ing countries, particularly in the event of TiLV listing by 2017). Whether such variations exist in other aquatic the OIE and where there is no method to differentiate the viruses such as TiLV remains to be determined and is of vaccine strain and wild strain (e.g. through sequencing). importance in terms of surveillance and control efforts. Depending on the efficacy of the vaccine, there may be an Given the importance of tilapia as a protein source in additional risk that vaccinated fish may become subclini- parts of the world, TiLV-associated losses may constitute a cally infected and contributing to the inadvertent spread of significant risk to household incomes and food security. the agent. Such issues need to be considered prior to the Socio-economic impact assessments should be encouraged instigation of mass vaccination programmes. It may be rel- to quantify the current or expected impact of disease as a evant to consider whether the use of autogenous inacti- result of infection with TiLV. It seems impossible to expand vated vaccines (single or polyvalent), with or without tilapia farming with the widely held belief that tilapia, adjuvants, should be promoted at large in countries and whether of nonimproved- or improved strains, is resistant areas where other approaches are unrealistic. to diseases. Basic principles of health management and Current information suggests that stress (e.g. transporta- biosecurity should be considered at all levels to ensure sus- tion and stocking of fingerlings and juveniles) may be an tainability of the tilapia industry. There must be focused important factor for the development of clinical outbreaks, attention to the development and implementation of prac- and management practices should aim to minimize the tical, affordable and effective BMPs to reduce disease and effect of transportation and handling. Good management environmental impacts for small-holder farmers. Effective practices (GMPs) of rapid removal of moribund and dead disease management is a shared responsibility and interna- fish should be encouraged together with the safe disposal of tional cooperation and productive alliances of govern- removed fish. The technology for producing specific patho- ments, industry and the community will be required to gen-free (SPF) stocks is available and it would be possible accomplish this goal. Future disease control programmes to establish SPF broodstock also for TiLV. While requiring should harness the potential of information technology a highly sensitive, nonlethal sampling methods for screen- tools (e.g. data mining, machine learning, image recogni- ing and selection of SPF broodstock and offspring, the tion, GIS mapping and predictive modelling) combined method could provide TiLV-free stocks to reduce the with novel surveillance and diagnostic technologies to

Reviews in Aquaculture, 1–15 12 © 2018 The Authors Reviews in Aquaculture Published by John Wiley & Sons Australia, Ltd Tilapia lake virus threat to tilapia industry accomplish provision of real-time solutions and early warn- Bacharach E, Mishra N, Briese T, Zody MC, KembouTsofack JE, ing systems to a diverse array of stakeholders (e.g. farmers, Zamostiano R et al. (2016a) Characterization of a novel researchers, policymakers). Only then will the impact of orthomyxo-like virus causing mass die-offs of tilapia. MBio 7: serious aquatic animal diseases on aquaculture production, e00431–16. livelihoods and trade be minimized. Bacharach E, Mishra N, Briese T, Eldar A, Lipkin WI, Kuhn JH In summary, TiLV is a newly emerging viral pathogen (2016b) ICTV Taxonomic Proposal 2016.016a-dM.A.v2.Tilapi- that poses a potential threat to the global tilapia industry. nevirus. Create the Unassigned Genus Tilapinevirus. [Cited 20 As current knowledge is still limited, there are several Dec 2017.] Available from http://www.ictv.global/proposals- important knowledge gaps that remain to be filled. To 16/2016.016a-dM.A.v2.Tilapinevirus.pdf Baldock FC, Blazer V, Callinan R, Hatai K, Karunasagar I, limit the negative impact and to prevent further spread Mohan CV et al. (2005) Outcomes of a short expert consulta- of the virus, combined approaches are required. tion on epizootic ulcerative syndrome (EUS): re-examination National- and international biosecurity efforts, effective of causal factors, case definition and nomenclature. In: BMPs, capacity building and widespread collaboration Walker P, Lester R, Bondad-Reantaso MG (eds) Diseases in between international and national stakeholders must be Asian Aquaculture V, pp. 555–585. Fish Health Section, Asian prioritised. Such strategies will aid the management and Fisheries Society, Manila. control efforts aimed at tackling TiLV while simultane- Behera BK, Pradhan PK, Swaminathan TR, Sood N, Paria P, Das ously aiding preparedness for rapid response to other A et al. (2018) Emergence of tilapia lake virus associated with emerging diseases in the future. mortalities of farmed Nile tilapia Oreochromis niloticus (Lin- naeus 1758) in India. Aquaculture 484: 168–174. Bowers RM, Lapatra SE, Dhar AK (2008) Detection and quanti- Acknowledgement tation of infectious pancreatic necrosis virus by real-time This work was undertaken as part of the CGIAR Research reverse transcriptase-polymerase chain reaction using lethal Program on Fish Agri-food Systems (FISH) in collabora- and non-lethal tissue sampling. Journal of Virological Methods 147 – tion with King Mongkut’s University of Technology Thon- : 226 234. buri (KMUTT) and the Norwegian Veterinary Institute. 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